Cell division in bacteria requires the concerted action of a large number of proteins that form a complex called the septal ring. Major challenges for the future include identifying all of these proteins and determining what they do on a biochemical level. Our long-term goal is to identify and characterize previously overlooked bacterial proteins that are important for cell division. Here we focus on a new class of cell division proteins that contain a ?SPOR? domain. SPOR domains are found in over 3000 proteins from over 1000 bacterial species, including many serious pathogens. SPOR domains bind septal peptidoglycan (PG) and thereby target many division proteins to the division septum. The specific form of PG to which SPOR domains bind is a ?denuded? glycan, a region of the amino sugar backbone of PG devoid of peptide side-chains. This unusual targeting mechanism sets SPOR domain proteins apart from the many septal ring proteins that localize via protein- protein interactions. This study will use four Escherichia coli SPOR domain proteins (DamX, DedD, FtsN and RlpA) to test two interrelated hypotheses: (a) different SPOR domains bind denuded glycans differently, and (b) these differences are physiologically important. In Aim 1 biolayer interferometry (BLI) and isothermal titration calorimetry (ITC) will be used to determine the affinity of SPOR domains for denuded glycans and the features of the glycan that are important for binding. In addition, NMR will be used to determine the first structure of a SPOR domain in complex with a PG ligand. In Aim 2, the four E. coli SPOR domain proteins will be examined in vivo using sophisticated single-molecule microscopy methods. Objectives of this aim include ascertaining whether different SPOR domain proteins localize to different sites at the septum and developing a more detailed picture of the overall architecture of the septal ring. Aim 3 will focus on the structure and function of DamX, using domain swapping and deletion analysis to identify important domains in that protein, ascribe those domains to specific functions and determine whether SPOR domains are functionally interchangeable. These experiments will connect the biochemistry and microscopy in Aims 1 & 2 to the actual function of SPOR domain proteins during cell division. Particular effort will be devoted to understanding the mechanism by which DamX inhibits cell division, because this is an unusual activity for a septal ring protein and may be relevant to pathogenesis by E. coli that infect the bladder.